During fiscal year 2011, we accomplished the following: 1. Completed studies on the chromosome conformation of the germline IgH locus in primary pro-B cells obtained from recombinase-deficient mice. These studies included 1) quantitative 3D-FISH to measure distances between different parts of the IgH locus in WT and Em-deficient cells, and 2) refinement of the bar-code FISH procedure to visualize chromosome conformation. 2. We used primary pro-B cells in which expression of the looping factor CTCF had been knocked down using shRNA-expressing retrovirus. We tested the predictions of our model derived from studies in #1 above. We found that Em-independent loops in the VH locus, that we predicted to be CTCF-dependent, were disrupted in CTCF-depleted pro-B cells. In contrast, Em-dependent loops, that were predicted to be YY1-dependent, were not disrupted in CTCF-depleted cells. These observations provide direct confirmation of the 2-step model for IgH locus chromosome conformation. 3. We tested several retrovirus shRNA constructs to identify one or more that efficiently depleted YY1 in cells. We found 2 out of 4 retroviruses depleted YY1 expression in mouse embryo fibroblasts. These viruses are currently being used to infect primary pro-B cells and pro-B cell lines, to down-regulate YY1 expression. YY1-depleted pro B cells will be used in 3D-FISH studies to test the complementary predictions of the 2-step model.